Adding new covalent bonds to proteins in live cells

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Project Summary/Abstract Proteins perform a variety of biological functions by interacting with different families of bimolecules. These interactions are largely noncovalent in nature. Although such noncovalent interactions provide diversity and dynamics for biology, their reversibility and weakness also impose strong limitations on researching and engineering protein-bimolecule interactions. In this application the natural barrier will be broken by designing and genetically incorporating bioreactive unnatural amino acids (Uaas) into proteins, which will enable proteins to bind with ribonucleic acids and carbohydrates in the covalent mode. The underlying innovation is to develop biocompatible chemistry, so that the Uaa will selectively react with the target ribonucleic acid or carbohydrate via proximity-enabled reactivity only upon binding, resulting in stable and irreversible linkage between protein and the target. To achieve these goals, new chemistries suitable for covalently targeting ribonucleic acids and carbohydrates in cellular and physiological conditions will be developed. The desired chemical functionality will be synthesized into the side chain of a Uaa, and the Uaa will be site-specifically incorporated into proteins in live cells via the genetic code expansion technology. This new covalent bonding strategy will be applied in live cells to understand RNA-protein recognition specificity, to identify substrate glycoproteins for glycosylation modifying enzymes, and to antagonize cell-cell communication. By harnessing the new covalent linkages inaccessible to natural proteins, this project will initiate a new dimension for researching and rationally engineering protein-RNA and protein-carbohydrate interactions. As such interactions are indispensable for biological functions and their aberrations are extensively implicated in various human diseases, new technologies resulting from this project will fundamentally impact both basic biological research and therapeutic applications.